RAB42 overexpression correlates with poor prognosis, immune cell infiltration and chemoresistance.

Background: RAB42 (Ras-related protein 42) is a new small GTPase that controls the vesicular trafficking from endosomes to trans-Golgi network in mammalian cells. However, the role of RAB42 in multiple cancers, especially in liver hepatocellular carcinoma (LIHC), has not been well investigated. Methods: A variety of cancer-related databases and online tools, including TCGA, GTEx, TARGET, QUANTISEQ, EPIC, RNAactDrug, CTR-DB, TIMER algorithms and Sangerbox, were applied to explore the correlation of RAB42 expression with prognosis, immune microenvironment, immune regulatory network, RNA modification, pathway activation and drug sensitivity in pan-cancer. The prognostic, immunomodulatory and tumor-promoting effects of RAB42 were verified in various malignancies and determined by a series of in vitro cellular experiments. Results: RAB42 is significantly overexpressed in most cancers with advanced pathological stages. Its overexpression is correlated with poor survival in pan-cancer. RAB42 overexpression has a high diagnostic accuracy of various cancers (AUC > 0.80). RAB42 overexpression not only correlates with distinct stromal immune infiltration and level of immune checkpoint molecules, but also associates with weak immune cell infiltration, immunomodulatory genes expression, and immunotherapeutic response to immune checkpoint inhibitors (ICIs). Additionally, RAB42 overexpression correlates with enhanced expression of m6A RNA methylation-related genes (MRGs) and its interactors. Moreover, overexpression of RAB42 serves as a drug-resistant marker to certain chemotherapies and acts as a potential biomarker for LIHC. Notably, RAB42 overexpression or activation promotes the cellular proliferation, migration and invasion of LIHC. Conclusion: Overexpressed RAB42 serves as a potential prognostic biomarker and therapeutic target in pan-cancer, especially in LIHC.

Effect of surface modification on the distribution of magnetic nanorings in hepatocellular carcinoma and immune cells.

Magnetic nanomaterial-mediated magnetic hyperthermia is a localized heating treatment modality that has been applied to treat aggressive cancer in clinics. In addition to being taken up by tumor cells to function in cancer therapy, magnetic nanomaterials can also be internalized by immune cells in the tumor microenvironment, which may contribute to regulating the anti-tumor immune effects. However, there exists little studies on the distribution of magnetic nanomaterials in different types of cells within tumor tissue. Herein, ferrimagnetic vortex-domain iron oxide nanorings (FVIOs) with or without the liver-cancer-targeting peptide SP94 have been successfully synthesized as a model system to investigate the effect of surface modification of FVIOs (with or without SP94) on the distribution of tumor cells and different immune cells in hepatocellular carcinoma (HCC) microenvironment of a mouse. The distribution ratio of FVIO-SP94s in tumor cells was 1.3 times more than that of FVIOs. Immune cells in the liver tumor microenvironment took up fewer FVIO-SP94s than FVIOs. In addition, myeloid cells were found to be much more amenable than lymphoid cells in terms of their ability to phagocytose nanoparticles. Specifically, the distributions of FVIOs/FVIO-SP94s in tumor-associated macrophages, dendritic cells, and myeloid-derived suppressor cells were 13.8%/12%, 3.7%/0.9%, and 6.3%/1.2%, respectively. While the distributions of FVIOs/FVIO-SP94s in T cells, B cells, and natural killer cells were 5.5%/0.7%, 3.0%/0.7%, and 0.4%/0.3%, respectively. The results described in this article enhance our understanding of the distribution of nanomaterials in the tumor microenvironment and provide a strategy for rational design of magnetic hyperthermia agents that can effectively regulate anti-tumor immune effects.

Progress in oncolytic viruses modified with nanomaterials for intravenous application.

In oncolytic virus (OV) therapy, a critical component of tumor immunotherapy, viruses selectively infect, replicate within, and eventually destroy tumor cells. Simultaneously, this therapy activates immune responses and mobilizes immune cells, thereby eliminating residual or distant cancer cells. However, because of OVs' high immunogenicity and immune clearance during circulation, their clinical applications are currently limited to intratumoral injections, and their use is severely restricted. In recent years, numerous studies have used nanomaterials to modify OVs to decrease virulence and increase safety for intravenous injection. The most commonly used nanomaterials for modifying OVs are liposomes, polymers, and albumin, because of their biosafety, practicability, and effectiveness. The aim of this review is to summarize progress in the use of these nanomaterials in preclinical experiments to modify OVs and to discuss the challenges encountered from basic research to clinical application.

[Expression of Key Enzymes in Glucose Metabolism in Chronic Mountain Sickness and Its Correlation with Phenotype].

OBJECTIVE: To explore the pathogenesis of erythrocytosis by detecting the key enzymes of glucose metabolism and glucose transporter in bone marrow erythrocytes of chronic mountain sickness (CMS), and analyzing its correlation with hemoglobin. METHODS: Twenty CMS patients hospitalized in Qinghai Provincial People's Hospital from January 2019 to December 2020 were selected as CMS group. Twenty males with leukocyte count > 3.5×109/L who had accepted bone marrow aspiration and had normal result were taken as control group. The mRNA and protein expression of key enzymes and glucose transporter in glucose metabolism in bone marrow CD71+ erythrocytes were detected by real time qPCR and Western blot, respectively. Glucose, lactic acid and 2,3-diphosphoglycerate in the bone marrow supernatant and serum were tested by ELISA. The mRNA and protein expression of key enzymes and glucose transporter, glucose, lactic acid and 2,3-diphosphoglycerate of the two groups were compared. Pearson correlation was used to analyze the correlation between key enzymes, glucose transporter in glucose metabolism in bone marrow CD71+ erythrocytes and hemoglobin. RESULTS: The expression of HK2, GLUT1 and GLUT2 mRNA in the CMS group were higher than those in the control group (P<0.001), while the expression of HK1, OGDH and COX5B mRNA were not different. The expression of HK2, GLUT1 and GLUT2 protein in the CMS group were higher than those in the control group (P<0.05). The levels of glucose and lactic acid in the bone marrow supernatant and serum in the CMS group were not different from those in the control group, while the level of 2,3-diphosphoglycerate was higher (P<0.001). Both HK2 and GLUT2 proteins were positively correlated with hemoglobin (r=0.511, 0.717). CONCLUSION: CMS patients may increase glycolysis by increasing the expression of HK2, and promote the utilization of glucose through high expression of GLUT1 and GLUT2 to meet the need of energy supply.

Different expression of circulating microRNA profile and plasma SP-D in Tibetan COPD patients.

COPD is the fourth leading cause of mortality, and is predicted to be the third leading cause of death worldwide by 2020. But few studies on Tibetan COPD of China. This study identifies distinctive miRNA signatures in Tibetan COPD patients from Tibetan healthy subjects that could serve as diagnostic biomarkers or describe differential molecular mechanisms with potential therapeutic implications. In this study, a total of 210 differentially expressed miRNAs were screened. Analysis of the functions of target genes of differentially expressed miRNAs via GO enrichment analysis revealed that they mainly influenced guanyl-nucleotide exchange factor activity, cell morphogenesis and the positive regulation of GTPase activity. KEGG pathway enrichment analysis showed that these target genes were mainly enriched in signaling by NGF, Axon guidance, developmental biology, ubiquitin mediated proteolysis, and PDGF signaling pathways. MiR-106-5p and miR-486-5p expression was validated in the complete cohort. Age, plasma miR-106-5p, miR-486-5p, SP-D protein levels, and SP-D mRNA level were also determined to be correlated with FEV1%Pred, and may as the risk factors of Tibetan COPD. The combination of plasma miR-106-5p, miR-486-5p and SP-D mRNA expression may be the best model to assist the diagnosis of Tibetan COPD.

Bone mesenchymal stem cell-derived exosomal microRNA-7-5p inhibits progression of acute myeloid leukemia by targeting OSBPL11.

BACKGROUND: Acute myeloid leukemia (AML) is a malignant clonal disease of hematopoietic stem- and progenitor-cell origin. AML features massive proliferation of abnormal blasts and leukemia cells in the bone marrow and the inhibition of normal hematopoiesis at onset. Exosomes containing proteins or nucleic acids are secreted by cells; they participate in intercellular communication and serve as key modulators of hematopoiesis. The purpose of this study was to investigate the effects of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) on the regulation of AML and the underlying mechanisms mediated by microRNA (miRNA). METHODS: Dysregulated miR-7-5p in AML patients was identified using qRT-PCR and its clinical significance was explored. Bioinformatic analysis revealed the target gene OSBPL11 that could be regulated by miR-7-5p. The findings were validated using a dual-luciferase reporter assay and western blotting. The functional genes of the PI3K/AKT/mTOR signaling pathway were identified, and the functional significance of miR-7-5p in AML cells was determined using a functional recovery assay. AML cells were co-cultured with exosomes originating from BMSCs overexpressing miR-7-5p to determine cell-cell regulation by Exo-miR-7-5p, as well as in vitro and in vivo functional validation via gain- and loss-of-function methods. RESULTS: Expression of miR-7-5p was decreased in AML patients and cells. Overexpression of miR-7-5p curbed cellular proliferation and promoted apoptosis. Overexpression of OSBPL11 reversed the tumorigenic properties of miR-7-5p in AML cells in vitro. Exo-miR-7-5p derived from BMSCs induced formation of AML cells prone to apoptosis and a low survival rate, with OSBPL11 expression inhibited through the PI3K/AKT/mTOR signaling pathway. Exo-miR-7-5p derived from BMSCs exhibited tumor homing effects in vitro and in vivo, and inhibited AML development. CONCLUSIONS: Exo-miR-7-5p derived from BMSCs negatively regulates OSBPL11 by suppressing the phosphorylation of the PI3K/AKT/mTOR signaling pathway, thereby inhibiting AML proliferation and promoting apoptosis. The data will inform the development of AML therapies based on BMSC-derived exosomes.

Expression profile of miRNA in NSCLC tissues in middle-altitude area.

Micro ribonucleic acid (miRNA) expression profile in non-small cell lung cancer (NSCLC) tissues in middle-altitude area was analyzed using the Affymetrix chip technique, to predict the target genes of abnormally-expressed miRNAs, and to analyze the target gene-related signaling pathways and cell biological functions regulated by them. The difference in miRNA expression profile in NSCLC tissues was analyzed using the Affymetrix chip technique. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed for the verification of some differentially-expressed miRNAs. The genes predicted by at least 6 out of 12 commonly used prediction methods of miRNA target genes, based on miRWalk2.0, were considered as target genes. The functions of differentially-expressed miRNA target genes were analyzed via Gene Ontology (GO) enrichment analysis, and the main signaling pathways involving target genes were analyzed via Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. There was abnormal expression of miRNAs in NSCLC tissues in the middle-altitude area. There were 140,405 target genes predicted for differentially-expressed miRNAs. The GO enrichment analysis of the functions of the target genes of differentially expressed miRNAs revealed that they mainly influence the binding process of intracellular components to protein, the positive regulation of biological process and the regulation of metabolic process. Moreover, these target genes were mainly enriched in the immunity, gene expression, metabolism and signal transduction, among which signal transduction was enriched with the most genes. The expression levels of miRNA-139-5p and miRNA-150-5p in lung cancer group were lower than those in the control group. The expression of miRNAs in NSCLC tissues in the middle-altitude area is abnormal, and most miRNAs are downregulated.

[Effect of HIF-1α on Angiogenesis-Related Factors in K562 Cells].

OBJECTIVE: To explore the mechanisms of angiogenesis in chronic myeloid leukemia (CML) through detecting the levels of angiogenesis-related factors secreted from K562 cells after overexpression and interference of HIF-1α gene in K562 cells. METHODS: The K562 cells were transfected by lentiviruses carried and interfered HIF-1α gene, then the transtected K562 cells with carried and interfered with HIF-1α gene were enrolled in overexpression and interference groups respectively, at the same time the K562 cells transfected by the empty virus were enrolled in control group. The cells were harvested after culture for 72 hours under normoxid condition. The transfection efficient in 3 groups was detected by fluorescence microscopy; the mRNA expression of HIF-1α gene and angiogenesis-related factors was detected by RT-PCR; the concentration of angiogenesis-related factors in the caltured supernatant was detected by ELISA. RESULTS: The optimal MOI of K562 cells transfected with lentivirus was 10 and the transfection efficiency was about 50%. The positive rate of transfection after screening by puromycin was more than 90%. The mRNA expression of ANG-I, ANG-II, TGF-α and VEGF in the interference group was lower than that in the over-expression group, and the TGF-ß1 mRNA expression in the interference group was higher than in the over-expression group. The mRNA expression of ANG-I and VEGF in the interference group was lower than that in the control group. TGF-αdid not could be detected, and the culture supernatant concentration of ANG-I and TNF-α in the interference group was lower than in the over-expression group, while the VEGF concentration in the interference group was higher than that in the over-expression group. All of the above-mentioned differences were statistically significant (P＜0.05). CONCLUSION: The positive K562 cells transfected with leutivirus have been harvested by screening with puromycin. The HIF-1α mRNA positively regulates the mRNA expression of ANG-1, ANG-2, TGF-α, VEGF in K562 cells, promotes the antocrine ability of ANG-1 and TNF-α, moreover not stimulates the autocrine of TGF-α, the up-regulation of HIF-1α expression can inhibit the expression TGF-ß1 in K562 cells and the autocrine of TGF-ß1.

[Significance of Bone Marrow Microvessel Density and Vascular- Related Factors in Multiple Myeloma].

OBJECTIVE: To investigate the clinical significance of bone marrow microvessel density(MVD) and angiogenesis related factors in multipic myeloma(MM). METHODS: Twenty cases of MM and 20 cases of simple fracture were selected and enrolled in MM group and control group respectively. The clinical data and results of laboratorial tests were collected; the bone marrow MVD of patients was detected by using the modified plastic-embedded pathologic sections of bone marrow tissue and histochemistry staining, the expression levels of amgiogenesis-related factors including VEGF, TNF-α, HGF, TGF-α, TGF-ß1, bFGF, Ang-Ⅰ, Ang-Ⅱ in bone marrow supernatant were detected by ELISA; the mRNA expression levels of above-mentioned cytokines in bone marrow mononuclear cells were detected by real time-PCR; the pearson correlation analysis was used to analyze the correlation of MVD with VEGF, HGF and bFGF levels. RESULTS: The MVD in MM group was significantly higher than that in control group (P＜0.001); the mRNA expression of VEGF, TGF-α, TGF-ß1 and HGF in bone marrow mononuclear cells of MM group was higher than that of control group(P＜0.001); the levels of VEGF, HGF, bFGF and THF-α in bone marrow supernatant of MM group were higher than those in control group(P＜0.05), moreover, the MVD positively correlated with levels of VEGF, HGF and bFGF in bone marrow(r=0.488, 0.472 and 0.457). CONCLUSION: The MVD and levels vessel-related factors in bone marrow supernatant of MM patients increase, among which the levels of VEGF and HGF in bone marrow supernatant are consistant with those mRNA expression level in bone marrow mononuclear cells, moreover, the MVD possitively cerrelates with levels of VEGF, HGF and bFGF in bone marrow supernatant, suggesting that the changes of bone marrow microenvironment vassel-related factors play an important role in angiogenesis and pathogenesis of multiple myeloma.

PI3K/Akt signaling transduction pathway, erythropoiesis and glycolysis in hypoxia (Review).

The purpose of this review is to summarize the research progress of PI3K/Akt signaling pathway in erythropoiesis and glycolysis. Phosphatidylinositol­4,5­bisphosphate 3­kinase (PI3K) is activated by numerous genes and leads to protein kinase B (Akt) binding to the cell membrane, with the help of phosphoinositide­dependent kinase, in the PI3K/Akt signal transduction pathway. Threonine and serine phosphorylation contribute to Akt translocation from the cytoplasm to the nucleus and further mediates enzymatic biological effects, including those involved in cell proliferation, apoptosis inhibition, cell migration, vesicle transport and cell cancerous transformation. As a key downstream protein of the PI3K/Akt signaling pathway, hypoxia­inducible factor (HIF)­1 is closely associated with the concentration of oxygen in the environment. Maintaining stable levels of HIF­1 protein is critical under normoxic conditions; however, HIF­1 levels quickly increase under hypoxic conditions. HIF­1α is involved in the acute hypoxic response associated with erythropoietin, whereas HIF­2α is associated with the response to chronic hypoxia. Furthermore, PI3K/Akt can reduce the synthesis of glycogen and increase glycolysis. Inhibition of glycogen synthase kinase 3ß activity by phosphorylation of its N­terminal serine increases accumulation of cyclin D1, which promotes the cell cycle and improves cell proliferation through the PI3K/Akt signaling pathway. The PI3K/Akt signaling pathway is closely associated with a variety of enzymatic biological effects and glucose metabolism.

Regulation of angiogenic factors by the PI3K/Akt pathway in A549 lung cancer cells under hypoxic conditions.

The aim of the present study was to investigate the influence of hypoxia and PI3K inhibition on angiogenic factors in A549 lung cancer cells. A549 cells were treated with the PI3K inhibitor LY294002 under normoxic and hypoxic conditions. Untreated cells were used as the control group and those treated by the inhibitor, as the suppression group. The cells were further divided based on normoxic or hypoxic conditions and named: Normoxic control group, normoxic suppression group, hypoxic control group and hypoxic suppression group. Expression levels of hypoxia-inducible factor (HIF)-1α and AKT1 mRNA in all groups were determined by reverse transcriptase-quantitative polymerase chain reaction and concentrations of vascular endothelial growth factor (VEGF), angiotensin II (ANG-II), transforming growth factor (TGF)-α/ß1, and tumor necrosis factor (TNF)-α in the culture supernatant were measured by enzyme-linked immunosorbent assay. The expression levels of HIF-1α and AKT1 mRNA in the hypoxic control group were higher than those in the normoxic control group and the expression levels of HIF-1α and AKT1 mRNA in the hypoxic control group were higher than those in the hypoxic suppression group. Compared to the normoxic control and normoxic suppression groups, the concentrations of VEGF and TNF-α in supernatant were higher in the hypoxic control and hypoxic suppression groups, respectively. However, TGF-α and TGF-ß1 demonstrated the opposite trend of the aforementioned factors. The concentration of ANG-II in the hypoxic suppression group was higher than that in the normoxic suppression group. In addition, compared to the normoxic control group and hypoxic control group, the concentrations of VEGF and TGF-ß1 in supernatant were lower in the normoxic suppression group and in the hypoxic suppression group, respectively. In conclusion, the results of the present study suggest that hypoxia can stimulate A549 lung cancer cells to secrete VEGF and TNF-α and inhibit TGF-α and TGF-ß1. The ability of A549 cells to secrete VEGF and TGF-ß1 is regulated by PI3K/Akt, and ANG-II expression may be regulated by the PI3K/Akt pathway under hypoxic condition.

MicroRNA-363 and GATA-1 are regulated by HIF-1α in K562 cells under hypoxia.

The aim of the present study was to investigate regulatory relationships among hypoxia-inducible factor-1α (HIF-1α), microRNA and erythroid transcription factors. K562 cells were transfected with HIF-1α knockout or with overexpression lentivirus of plasmid (MOI 10). The cells were divided into 3 groups: the negative control, overexpressing and interference groups. The cells were cultured under normoxia and hypoxia. Expression of miR-17*, miR-363 and miR-574-5p in the three groups was determined by quantitative PCR. Expression levels of erythroid transcription factor mRNAs such as GATA-1/GATA-2 and nuclear factor­erythroid 2 (NF-E2) were measured using RT-qPCR while the protein expression was studied using western blot analysis. Under normoxia or hypoxia, the levels of miR-17*, miR-363 and miR­574-5p in the overexpression group were higher than those in the other groups. Differences were statistically significant (P<0.05). Under hypoxia, the level of miR-363 in the interference group was less than that in the negative control group and difference was statistically significant (P<0.05). The level of GATA-1 mRNA in the overexpression group was higher than that in the negative control group, however, in the interference group the level was lower than that in the overexpression group under both normoxic and hypoxic conditions. The level of GATA-2 mRNA in the interference group was higher than that in other two groups under normoxic or hypoxic conditions. The NF-E2 mRNA was reversely related to GATA-2. The levels of HIF-1α, GATA-1 and NF-E2 mRNAs in the negative control under hypoxia were higher than those of normoxia. The level of HIF-1α mRNA in the overexpression group in hypoxia was lower than that in normoxia, while the GATA-1 and GATA-2 mRNA showed a reverse association. The levels of HIF-1α and GATA-2 mRNA in the interference group under hypoxia were higher compared to those of normoxia. Differences were statistically significant (P<0.05). Western blot results suggested that GATA-1, GATA-2 and NF-E2 protein expression correlated with changes in their respective mRNA transcription levels. The results therefore suggested that GATA-l and miR-363 were involved in the regulation of hematopoiesis via the HIF-1α pathway in K562 cells under hypoxic condition. The hsa-miR-17* and hsa-miR­574-5p were not entirely dependent on HIF-1α, suggesting possible complex regulatory mechanisms involved in hypoxia.